Thymoquinone from Nigella sativa Seeds Promotes the Antitumor Activity of Noncytotoxic Doses of Topotecan in Human Colorectal Cancer Cells in Vitro

Abstract

Topotecan, a topoisomerase I inhibitor, is an anticancer drug widely used in the therapy of lung, ovarian, colorectal, and breast adenocarcinoma. Due to the primary dose-limiting toxicity of topotecan, which is myelosuppressive, it is necessary to identify other chemotherapeutic agents that can work synergistically with topotecan to increase its efficacy and limit its toxicity. Many studies have shown synergism upon the combination of topotecan with other chemotherapeutic agents such as gemcitabine. Other studies have demonstrated that pre-exposing cells to naturally occurring compounds such as thymoquinone, followed by gemcitabine or oxaliplatin, resulted in higher growth inhibition compared to treatment with gemcitabine or oxaliplatin alone. Our aim was to elucidate the underlying mechanism of action of topotecan in the survival and apoptotic pathways in human colon cancer cell lines in comparison to thymoquinone, to study the proapoptotic and antiproliferative effects of thymoquinone on the effectiveness of the chemotherapeutic agent topotecan, and to investigate the potential synergistic effect of thymoquinone with topotecan. Cells were incubated with different topotecan and thymoquinone concentrations for 24 and 48 hours in order to determine the IC50 for each drug. Combined therapy was then tested with ± 2 values for the IC50 of each drug. The reduction in proliferation was significantly dose- and time-dependent. After determining the best combination (40 µM thymoquinone and 0.6 µM topotecan), cell proteins were extracted after treatment, and the expression levels of B-cell lymphoma 2 and of its associated X protein, proteins p53 and p21, and caspase-9, caspase-3, and caspase-8 were studied by Western blot. In addition, cell cycle analysis and annexin/propidium iodide staining were performed. Both drugs induced apoptosis through a p53-independent mechanism, whereas the expression of p21 was only seen in thymoquinone treatment. Cell cycle arrest in the S phase was detected with each compound separately, while combined treatment only increased the production of fragmented DNA. Both compounds induced apoptosis through the extrinsic pathway after 24 hours; however, after 48 hours, the intrinsic pathway was activated by topotecan treatment only. In conclusion, thymoquinone increased the effectiveness of the chemotherapeutic reagent topotecan by inhibiting proliferation and lowering toxicity through p53- and Bax/Bcl2-independent mechanisms.

Thymoquinone as a Potential Adjuvant Therapy for Cancer Treatment: Evidence from Preclinical Studies

“…In addition to the induction of synergistic cytotoxic effects through interference in tumor growth and survival signaling, combination treatment with TQ also exhibited chemo-protective effects and limited organ toxicity (Brown et al, 2014). Numerous other studies have investigated the potential chemo-potentiating effects of TQ with diverse classes of chemotherapeutic drugs (summarized in Table 1) including anti-estrogen (e.g., tamoxifen), topoisomerase-I inhibitor (e.g., topotecan), and microtubule disrupting agents (Rajput et al, 2013; Khalife et al, 2014, 2016; Dirican et al, 2015; Ganji-Harsini et al, 2016; Sakalar et al, 2016). Rajput et al recently reported that TQ can cause reversal of tamoxifen resistance in triple negative breast cancer cells by interfering in Akt-mediated induction of apoptosis inhibitory protein such as X-linked inhibitor of apoptosis protein (XIAP; Rajput et al, 2013).…”

North African Medicinal Plants Traditionally Used in Cancer Therapy

“…It was also cytotoxic toward A549 lung carcinoma and DLD-1 colon adenocarcinoma cells (IC 50 = 13 and 5.9 μM, respectively; Bourgou et al, 2010). Khalife et al (2016) described that the cytotoxic effect was dose- and time-dependent (IC 50 = 59.2 and 68.4 μM, for 24 and 48 h, respectively) for HT-29 colorectal carcinoma cells. Woo et al (2011) described the cytotoxic effect of thymoquinone against different breast cancer lines with MDA-MB-231 cells as the most susceptible ones (IC 50 = 11 μM after 48 h).…”

“…Thus, only studies with its major compound (thymoquinone) will be referred. Thymoquinone was able to cause cell cycle arrest at S phase (Khalife et al, 2016). By contrast, Woo et al (2011) described that this compound caused accumulation of cells in the sub-G1 phase without effect on p53 activation.…”

5,6-Dihydropyrrolo[2,1-<i>a</i>]isoquinolines as Alternative of New Drugs with Cytotoxic Activity

“…The cytotoxic activities of our most active compounds 4d and 4j were compared with those of the commercially available Topotecan, 16) Irinotecan, 17) Etoposide, 18) Tamoxifen, 19) and Cisplatin. 20) As shown in Table 3, compound 4d (3-OH) was ten times more active than cisplatin in the prostate PC-3 cell line, almost two thousand times more active than irinotecan in the colon HCT-15 cell line.…”